DK144033B - METHOD OF ANALOGUE FOR THE PREPARATION OF L-3- (3,4-DIHYDROXY-PHENYL) -2-METHYL-ALANINE PEPTIDES OR PHARMACOLOGICAL ACCEPTABLE SALTS THEREOF - Google Patents

Description

(19) DENMARK

| p (12) PUBLICATION OR 14 ^ + 033 B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 1038/76 (51) IntCI. * C 07 C 103/62 (22) Filing date 10 Mar. 1976 (24) Race day 10 Mar 1976 (41) Aim. available 13 · S ep. 1976 (44) Presented 23 Nov. 198I (86) International Application No. - (86) International Filing Day - (85) Continuation Day - (62) Master Application No. -

(30) Priority Mar 12 1975 * 2510634, DE

(71) Applicant BOEHRINGER MANNHEIM GMBH, Mannhelm-Waldhof, DE.

(72) Inventor Franz Braun, DE: Kurt Stach, DE: Max Thiel, DE: Gis * hert Sponer, DE: Karl Dietmann, DE.

(74) Plenipotentiary Chaa. Hude.

(54) Analogous process for the preparation of L-3- (5β-dihydroxy-phenyl) -2-methyl-alanine peptides or pharmaceutically acceptable salts thereof.

The present invention relates to an analogous process for the preparation of novel derivatives of L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine peptides of the general formula X

HAY

) - \? H3 EO- ^ r V-ch2-c-cooh (i)

® \ = / NH-CO-A-NH-R

ISLAND

ISLAND

wherein A means a straight or branched alkylene chain having 1-5 car =? hydrogen atoms, which may be substituted by a hydroxy group or by an optionally 1-2 hydroxy groups bearing phenyl group, and R represents a hydrogen atom, an alkyl group having 1-3 carbon atoms, a glycyl group or an alanyl group, or pharmacologically acceptable salts thereof.

144033 2

All carbon atoms in groups A and R with an asymmetric center may have L or D configuration, with the L configuration being preferred. The present invention relates to both the racemic mixtures and the pure L and D enantiomers.

As the alkyl group R having 1-3 carbon atoms, the methyl group is preferred.

The novel compounds and their pharmacologically acceptable salts have blood pressure lowering properties.

The process is characterized by condensing L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine of the general formula II

H0 \ _ ch3 H0-ff ch2-c-cooh (II) nh2

if hydroxy and / or carboxy groups may be temporarily protected, with carboxylic acids of general formula III

HOOC-AZ (III) or reactive derivatives thereof, wherein A has the meaning given above and Z means a reactive group or the group -NHR, wherein R has the above meaning, wherein, if desired, a hydroxy group present in the alkylene group A and -NHR- the group may be temporarily protected, and in the case that Z represents a reactive group, then this group replaces with the grouping -NHR and then optionally decomposes protecting groups in a manner known per se and, if desired, converts the compounds thus obtained to their pharmacologically acceptable salts.

The preparation of the compounds of the general formula I is carried out according to the commonly used methods in peptide chemistry.

3 144033

Preferably, in the presence of inert solvents (e.g., dioxane) and water-scavenging agents (e.g., dicyclohexylcarbo = diimide) are employed at room temperature.

As temporary protecting groups for the hydroxy substituents of the compounds of general formulas II and III, the usual hydroxy protecting groups, e.g. the benzyl or benzyloxycarbonyl = group.

For example, the carboxy group of L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine of formula II can be protected by esterification, preferably with a lower aliphatic alcohol or benzyl alcohol.

The temporary protection of the -NHR grouping can be done by the usual amino protecting groups, e.g. using the b enzyloxycarbonyl group.

As reactive derivatives of carboxylic acids of general formula III, acid halides, acid anhydrides or carboxylic acid esters are preferably used.

Reactive groups Z in compounds of the general formula III are especially acid residues, e.g. of halohydric acids and sulfonic acids.

The present compounds of general formula I have amphoteric character. They can therefore form pharmacologically acceptable salts with both acids and bases. For the preparation of acid addition salts, the subject compounds, preferably in an organic solvent, are reacted with the equivalent amount of a pharmacologically acceptable organic or inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, acetic acid, citric acid, maleic acid and benzoic acid. The neutralization of the carboxyl group may e.g. is carried out by reaction with alkali metal and alkaline earth metal bases, with ammonia or with organic amines.

For the preparation of drugs, the compounds I are mixed in a manner known per se with suitable pharmaceutical carriers, flavoring, flavoring and coloring agents and are formed, for example, as tablets or suspended or dissolved with the addition of suitable excipients in water or oil, e.g. olive oil.

4 144033

The compounds of the invention were tested in a series of tests to determine their effectiveness as antihypertensive agents.

The following test methods were used for the test.

The test animals were rats in which arterial catheters via the femoral artery had been implanted into the aorta by a sterile operation. It was possible to measure the animals' blood pressure in the waking state directly in the blood with a transducer (Statham Transducer, Type TP 23 D6) via a carrier frequency measurement bridge. The animals were treated by administration of 10% common salt (sodium chloride) in their food, and as of the 6th week of their life, 5 mg per week was injected twice a week. times of II-deoxycor = ticosterone acetate per animals, thereby developing an arterial high pressure with values from 190 to 130 mm Hg on each side of the mean.

The test compounds were administered to the animals as follows. After blood pressure control values were determined, the animals received the test compound orally suspended in 10 ml of 1% methyl = cellulose solution at a dose of 0.6 mmol / kg, and measurements were taken 4 hours after oral administration of the compound.

The compounds are listed in the table below. The values given in the table in each case represent the mean of at least 6 individual measurements of the reduction in blood pressure (in mm Hg) per day. connection given.

Table

Blood Pressure Reduction

Active Compound Hg-α-methyl-dopa 33 ^ 4.9 L-alanyl-L-alanyl-L-3- (3,4-dihydroxy + phenyl) -2-methyl-alanine 58-5.8 L-alanyl L-3- (3,4-dihydroxy-phenyl) - + 2-methyl-alanine 51-6.0 L-seryl-L-3- (3,4-dihydroxy-phenyl) - + 2-methyl-alanine 52 -7.5

Glycyl-L-3- (3,4-dihydroxy-phenyl) -2- + methyl-alanine 37-9.1

Control (1% methylcellulose) -4-2.5 (increase) 5 144033

In addition to the compounds mentioned in the following examples, preferred compounds in the sense of the invention are the following compounds:

Glycyl-glycyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine.

In the following examples, the present compounds and the process of the invention are illustrated for their preparation. The following abbreviations are used: DC = Thin-layer chromatography DCC = NjN'-dicyclohexylcarbodiimide

Cbo = Benzyloxycarbonyl THF = Tetrahydrofuran.

Example 1.

Glycyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine.

Variant I: 11.25 g (0.05 mol) of L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine-methyl = ester (m.p. 164-166 ° C, [oc] = -6.3 ° (c = 1, in 1N HCl)) is suspended in 250 ml of absolute dioxane in a 500 ml three-necked flask fitted with a stirrer and chloro-calcium tube. In succession, 12.52 g (0.06 mole) of benzyloxycarbonyl-glycine and 10.3 g (0.05 mole) of N, N'-dicyclohexylcarbodiimide (DCC) are added and stirred at room temperature under moisture exclusion. After only a few minutes, N, N'-dicyclohexylurea begins to crystallize.

After a further 4 hours stirring and 16 hours standing at room temperature, the urea (10.0 g, 89.4 $!) Is suctioned off and the filtrate is evaporated in vacuo. The highly viscous residue is taken up in 150 ml of vinegar ester and washed successively four times with 20 ml of approx. 5% sodium hydrogen carbohydrate = night solution, four times with 20 ml of 1N hydrochloric acid and then twice with 30 ml of water. The vinegar ester phase containing the desired reaction product is evaporated after drying with sodium sulfate in vacuo. 20.82 g (100.0% of theory) of benzyloxycarbonyl-glycyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester is obtained as a colorless amorphous powder which (tested thin layer chromatographically) ) is only slightly contaminated.

6 144033

For complete purification, the crude product can be dissolved in 100 ml of chloroform-methanol mixture (15: 1) and chromatographed on a column (90 x 5.5 cm) with silica gel 60 (70 - 230 mesh, Merck) with the same solvent mixture. Yield 17.5 g (84.1% of theory) as colorless amorphous powder.

To hydrolyze the methyl ester group, add 10.4 g (0.025 mol) of benzyloxycarbonyl-glycyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester in a three-necked flask equipped with a stirrer. -drip funnel and gas supply. After the complete removal of the air by means of a weak nitrogen flow, 95 ml of 1 N sodium hydroxide solution is allowed to flow through the dropping funnel and the solution is stirred for 30 minutes at room temperature. Then 95 ml of 1N hydrochloric acid is added dropwise. The crude hydrolysis product separates as a very cool oil which is taken up in 100 ml of vinegar ester and extracted three times from the vinegar ester solution with 30 ml of 10% sodium bicarbonate solution for further purification. From the combined aqueous extract, the free carboxylic acid is separated by gentle acidification with 1N hydrochloric acid, absorbed for approx. 100 ml of vinegar ester and dried with sodium sulfate. After complete distillation of the solvent in vacuo, 9.0 g (89.5% of theory) of thin layer chromatographic (DC) pure benzyloxycarbonylglycyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine is obtained as a colorless , amorphous foam.

For hydrogenolysis of the benzyloxycarbonyl group (Cbo), 2.5 g of palladium oxide in 130 ml of methanol is prehydrogenated at room temperature under normal pressure. Then, the solution of 20.12 g (0.05 mol) of benzyloxy = carbonylglycyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine in 170 ml of methanol is added and hydrogenated at room temperature in the usual manner for approx. .

4 hours under hydrogen flow until carbon dioxide decomposition is complete. The catalyst is then suctioned off and the solvent is distilled off in vacuo. 13.45 g (100% of theory) of colorless amorphous crude product is obtained, which is taken up in 28 ml of water and cooled to 0 ° C. From the solution, 11.88 g of thin layer chromatographically and analytically pure glycyl L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine dihydrate crystallize with m.p. 187 ° C, [α] D -8.0 ° ( c = 1, in Hg0).

Variant II: In a 2 liter four neck flask with stirrer, 2 drip funnels, gas line, pH electrode and thermometer is added to 45 g (0.2 mole) of L-3- (3,4-dihydroxyphenyl) -2-methyl alanine methyl ester, 300 ml of water and 500 ml of chloroform, and cooled vigorously to 0 ° C. Then 12.34 g (0.11 mol) of chlorine = acetyl chloride dissolved in 12 ml of chloroform are added dropwise over 1 hour. After a further 45 minutes, a slight stream of nitrogen is allowed to air-displace and dropwise 14.8 g (0.13 mole) of chloroacetyl chloride dissolved in 15 ml of chloroform and a solution of 36.96 g (0.26 mole) of sodium carbonate in 118 ml of water 0 ° C over 1.25 hours (pH of solution 7.5 - 8.5). Stir for an additional 1.5 hours without cooling. The unreacted methyl ester (20.0 g = 44.4%) is then suctioned off and the chloroform solution is separated and dried.

33.0 g (54.8% of theory) of N-chloroacetyl-L-3- (3,4-dihydroxy = phenyl) -2-methyl-alanine methyl ester is obtained as a yellow viscous oil.

30.17 g (0.1 mole) of methyl ester are hydrolyzed in a nitrogen atmosphere in 305 ml of 1 N sodium hydroxide solution, then neutralized with approx. 77 ml of 4N hydrochloric acid and extract the separated oil twice with 150 ml of vinegar ester. The extract dried with sodium sulfate is evaporated in vacuo. 26.1 g (91.0% of theory) of N-chloroacetyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine are obtained as yellow oil.

28.7 g (0.1 mole) of N-chloroacetyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine are dissolved in a nitrogen atmosphere in 260 ml of concentrated ammonia and allowed to stand at room temperature for 5 days. turnover. Then, evaporate in vacuo and purify the crude column chromatographically (column 60 x 6.4 cm, silica gel 60, Merck, eluent: n-butanol-acetic acid-water 2: 1: 1). After evaporation in vacuo and recrystallization from 35 ml of water, the DC pure eluate results in 22.8 g (75.0% of theory) of glycyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine dihydrate, mp 186 -187 ° C.

Example 2.

L-alanyl-L-3- (5,4-dihydroxy-phenyl) -2-methyl-alanine.

11.25 g (0.05 mole) of L-3 * - (3,4-dihydroxy-phenyl) -2-methyl-alanine-methyl = ester and 13.38 g (0.06 mole) of benzyloxycarbonyl-L-alanine (m.p. 84-86 ° C, [cc] + -15.6 ° (c = 2, acetic acid)) is reacted with 10.3 g (0.05 mole) of DCC in the same manner as in Example 1, I indicated and worked up . 21.5 g (100% of theory) of colorless crude product is obtained, which after column chromatographic purification results in 16.51 g (76.9% of theoretically) pure benzyloxycarbonyl-L-alanyl-L-3- (3 4-dihydroxy-phenyl) -2-methyl-alanine, methyl ester.

10.75 g (0.025 mol) of benzyloxycarbonyl-L-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester are hydrolyzed as described in Example 1, with 95 ml of 1 N sodium hydroxide solution for 50 minutes at room temperature and worked up. 9.46 g (91.090 of theory) of thin layer chromatographically pure benzyloxycarbonyl-L-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine (colorless foam) are obtained.

20.8 g (0.05 mole) of benzyloxycarbonyl-L-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine are hydrogenated as described in Example 1.1 for 1 hour at room temperature to hydrogenolysis of the Cbo group and reprocessed. 13 »38 g (94.7% of theory) of crude product is obtained, which is recrystallized from 13.5. ml of water results in 11.7 g of analytically pure L-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine trihydrate, mp 82 - 87 ° C (in small fused tubes), [ α] 0 -26.9 ° (c = 1, in H2 O).

Example 3 · D-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine.

15.75 g (0.07 mol) of L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine-methyl = ester and 16.83 g (0.075 mol) of Cbo-D-alanine (m.p. 84 - 86) ° C, [a] § + 14.8 ° (c = 2, acetic acid)) is reacted in 350 ml of dioxane with 14.4 g (0.07 mol) of DCC as set forth in Example 1, I and then worked up. 30.1 g (100% of theory) of colorless crude product is obtained. The column chromatographic purification results in 29.1 g (96.6%) of DC pure benzyloxy = carbonyl-D-alanyl-L-3- (3,4-dihydroxyphenyl) -2-methyl-alanine methyl = ester as colorless powder .

10.75 g (0.025 mol) of Cbo-D-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester are hydrolyzed as described in Example 1, with 95 ml of 1 N sodium hydroxide solution for 2 hours at room temperature and worked up. 9.46 g (91.0% of theory) of DC-pure Cbo-D-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine are obtained as colorless foam.

The hydrogenolysis of the Cbo group is carried out with 20.8 g (0.05 mol) of Cbo-D-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine as described in Example 1, In, and that is worked up. 14.05 g ($ 99.6 of theory) of crude product is obtained, which is recrystallized from ca. 20 ml of water. Yield 8.35 g of colorless, analytically pure D-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine monohydrate, m.p. 205 ° C, [α] 21 + 33.7 ° (c = 1, in H₂O).

Example 4

L-N-methyl-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine.

15.75 g (0.07 mol) of L-3- (5,4-dihydroxy-phenyl) -2-methyl-alanine-methyl = ester and 18.79 g (0.077 niol) of Cbo-LN-methyl-alanine ( oil, [α] 0 -24.8 ° (c = 1, in dimethylformamide)) is reacted in 350 ml of anhydrous dioxane with 14.42 g (0.07 mol) of DCC as described in Example 1, I and worked up then. 29.21 g ($ 94.0 of theory) of colorless crude product is obtained. The column chromatographic purification (eluent: chloro = form-methanol 20: 1) results in 14.18 g ($ 46.5 of theory) of DC-pure Cbo-LN-methyl-alanyl-L-3- (3,4-dihydroxy) -phenyl) -2-methyl-alanine methyl ester (amorphous powder).

11.1 g (0.025 mol) of CbO-LN-methyl-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester are hydrolyzed as in Example 1, I described for 1 hour at room temperature. and reprocessed. Yield 9.20 g (85.5% of theory) powdered DC pure Cbo-L-N-methyl-alanyl-L-3- (3,4-di = hydroxy-phenyl) -2-methyl-alanine.

The hydrogenolysis of the Cbo group is carried out with a solution of 10.27 g (0.025 mol) of Cbo-LN-methyl-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine in 90 ml of methanol and 30 ml of water, analogous to that of Example 1, with 1.25 g of palladium oxide in 100 ml of methanol, are worked up. 7.11 g (96.0 $ of theory) of crude product is obtained, which is purified by column chromatography (column: 90 x 3 cm, silica gel 60, 70 - 230 mesh, Merck; eluent: acetone-water 4: 1). The DC pure eluate is evaporated in vacuo, the residue is dissolved in a little water, slowly precipitated with ethanol and evaporated to dryness. There is thus obtained 5.69 g (76.8 $ of theory) of powdered LN-methyl-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine, which after drying at room temperature still contains 0 , 95 moles of water and 0.63 moles of ethanol, [cc] p + 38.9 ° (c = 1, in methanol).

Example 5

Β -alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine.

15.75 g (0.07 mol) of L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine-methyl = ester and 17.20 g (0.077 mol) of Cbo-β-alanine (m.p. 106 ° C) ) is reacted in 350 ml of dioxane with 14.4 g (0.07 mol) of DCC as set forth in Example 1, I and worked up. Yield of the crude product is 27.8 g (92.4% of theory) of colorless powder. The column chromatographic purification results in 15.15 g (50.3% of theory) of DC pure Cbo-β-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester.

10.75 g (0.025 mol) of Cbo-β-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester are hydrolyzed as indicated in Example 1, I for 1½ hours at room temperature and worked up . Yield 8.90 g (85.6% of theory) DC-pure Cbo-β-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine (colorless foam).

The hydrogenolysis of the Cbo group is carried out with a solution of 12.49 g (0.03 mol) of Cbo-β-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine in 110 ml of methanol and 40 ml of water and with 1 g of palladium oxide in 100 ml of methanol according to the example of Example 1. The suction catalyst is washed well with 200 ml of water and the filtrate is evaporated in vacuo. 7.88 g (92.9% of theory) of crystallized crude product, which is recrystallized from 100 ml of water, results in 6.64 g (78.4% of theory) of colorless, analytically pure β-η1θ 3- (3> 4-dihydroxy-phenyl) -2-methyl-alanine dihydrate, m.p. 147 ° C, [α] p -37.7 ° (c = 1, in acetic acid).

Example 6

L-seryl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine.

14.63 g (0.065 mol) of L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester and 22.39 g (0.068 mol) of N-benzyloxycarbonyl-O-benzyl-L-serine in 325 ml of dioxane to react with 13.36 g (0.065 mol) of DCC as set forth in Example 1, I and work up. Yield 34.5 g (100% of theory) colorless foam. The column chromatographic purification (column: 90 x 4.5 cm, silica gel 60 Merck; Eluant: chloroform-methanol 49: 1) results in 21.88 g (64.1% of theory) of DC-pure N-Cbo-O-benzyl -L-Seryl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine-methyl = ester as amorphous powder.

11.40 g (0.025 mol) of N-Cbo-O-benzyl-L-seryl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester is dissolved in 100 ml of THF and hydrolyzed as described in Example 1, I with 50 ml of 2N sodium hydroxide solution for 3 hours at 35 ° C. Then neutralize by approx. 50 ml of 2N hydrochloric acid and distill off THF completely in vacuo. After adding approx. 40 ml of water are taken up the crude hydrolysis product in 100 ml of vinegar ester and further worked up as indicated in Example 1, I. Yield 10.20 g (78.4% of theory) of DC-pure N-Cbo-O-benzyl-L seryl L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine (colorless powder).

The hydrogenolysis of the N-Cbo and O-benzyl group is carried out as described in Example 1, by For 6 hours at 25 ° C hydrogen passes through a solution of 13.06 g (0.025 mol) of N-Cbo-O-benzyl-L-seryl-L-3- (3,4-dihydroxy-phenyl) -2-methyl alanine in 150 ml dioxane and 25 ml 1N hydrochloric acid added fresh pre-hydrogenated palladium (1.25 g palladium oxide in 90 ml dioxane and 30 ml water). After suction of the catalyst, the filtrate is neutralized by approx. 25 ml of 1 N sodium hydroxide solution and evaporate to dryness in vacuo. The residue is dissolved in 70 ml of water and cooled to crystallize to 0 ° C. 2.24 g (30.1% of theory) of colorless, analytical and DC-pure L-seryl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine are obtained, which after drying in a vacuum exMatar still contains 2.3 moles of water; mp 196 - 197 ° C (decomposition), [oc] p® -4.4 ° (c = 1, in acetic acid).

Example 7 L-leucyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine.

As indicated in Example 1, I is subjected to 16.90 g (0.075 mole) of L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester and 21 »87 g (0.083 mole) of Cbo-L -leucine ([α] + -18.4 ° (c = 1, in acetic acid)) in 380 ml of dioxane peptide synthesis with 15.45 g (0.075 mol) of DCC. Yield 35.4 g (100% of theory) colorless powder. The column chromatographic purification (as in Example 1, I) results in 21.70 g (61.4% of theory) of DC pure Cbo-L-leucyl-L-3- (3,4-dihydroxy-phenyl) -2- methyl alanine methyl ester as amorphous powder.

18.88 g (0.04 mol) of Cbo-L-leucyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester are hydrolyzed as indicated in Example 1, I with 152 ml of 1 N sodium hydroxide solution. for 2 hours at room temperature and then worked up. 14.32 g (78.2% of theory) of DC-pure Cbo-L-leucyl-L-3- (3,4-12,440 dihydroxy-phenyl) -2-methylalanine are obtained as amorphous powder.

The cleavage of the Cho group is carried out as indicated in Example 1, I.

2.0 g of palladium oxide is dehydrogenated in 110 ml of methanol and 25 ml of water. To this is added the solution of 18.34 g (0.04 mol) of Cbo-L-leucyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine in 120 ml of methanol and 50 ml of water and hydrogenate. for 2 hours. After processing, 13.0 g (100% of theory) of crude product is obtained, which is recrystallized from 30 ml of water. Yield 8.34 g of colorless, DC and analytically pure L-leucyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine, which after drying in a vacuum desiccator contains 2.5 moles of water; mp 106 - 107 ° C (in small molten tubes), [α] 20 ° -21.4 ° (c = 1, in EL₂O).

Example 8.

L-phenylalanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine.

As in Example 1, I was subjected to 15.78 g (0.07 mol) of L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester and 23.92 g (0.08 mol) of Cbo -L-phenylalanine (m.p. 85 - 87 ° C, [α] p + 4.6 ° (c = 1.5, in acetic acid)) peptide synthesis in 350 ml dioxane with 14.42 g (0.07 mol) DCC . Yield 35.5 g (100% of theory) colorless foam. The column chromatographic purification (performed as set forth in Example 1, i) results in 33.67 g (95% of theory) of Cbo-L-phenyl-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl -alanine methyl ester as amorphous powder.

20.26 g (0.04 mol) of Cbo-L-phenylalanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester are hydrolyzed similar to Example 1, with 152 ml of 1 N sodium hydroxide solution in 3 hours at room temperature and work up. 16.35 g (83% of theory) of Cbo-L-phenylalanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine are obtained as a yellow, DC-pure powder.

19.70 g (0.04 mol) of Cbo-L-phenylalanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine is hydrogenated for hydrogenolysis of the Cbo group as set forth in Example 1, 5 hours at room temperature (2.0 g of palladium oxide in 23Ο ml of methanol) and work up. 14.08 g (98.1% of theory) of crude product is obtained. This material is precipitated from an alcoholic solution with ether and dried for 2 hours at 80 ° C in oil pump vacuum over diphosphorus pentoxide. Yield 9.61 g of pure L-phenylalanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine monohydrate, mp 160-163 ° C (decomposition), [α] + 8.1 ° (c = 1, in methanol).

13 144033

Example 9

L-Tyr oyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine.

9.0 g (0.04 mole) of L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine-methyl = ester and 22.08 g (0.063 mole) of N-Cbo-L-tyrosine dihydrate ([α] + + 3.8 ° (c = 1.5, in acetic acid)) is reacted in 200 ml of dioxane with 11.3 g (0.055 mol) of DCC and worked up as indicated in Example 1, I and column chromatographed. 19.7 g (94,290 of theory) of Cbo-L-tyrosyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester (colorless powder) are obtained.

20.90 g (0.04 mole) of N-Cbo-L-tyrosyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester is hydrolyzed according to Example 1, I with 192 ml IN baking soda for 2¾ hours at room temperature and work up. 18.20 g (89.5% of theory) of DC-pure Cbo-L-typosyl, L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine (colorless powder) are obtained.

The hydrogenolysis of the Cbo group is carried out as described in Example 1, I.

1.5 g of palladium oxide is dehydrogenated in 100 ml of methanol and 20 ml of water. To this is added a solution of 15.25 g (0.03 mol) of N-Cbo-L-typogyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine in 150 ml of methanol and 30 ml of water. , and hydrogenated for 3 hours. After workup, 10.4 g ($ 92.5 of theory) of colorless crude product is obtained, which is recrystallized from 20 ml of water. Yield 8.1 g ($ 72.6 of theory) DC and analytically pure L-tyrosyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine, m.p. 205 ° C (decomposition) by rapid heating), [α] + 6.5 ° (c = 1, in methanol).

Example 10.

L- [3- (3,4-dihydroxy-phenyl) -2-methyl-alanyl] -L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine 13.50 g (0.06 mol) L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine-methyl = ester and 22.80 g (0.066 mol) of N-Cbo-L-3- (3,4-dihydroxy-phenyl) ) -2-Methyl-alanine is reacted in 300 ml of dioxane with 12.36 g (0.06 mol) of DCC as set forth in Example 1, I and worked up. The column chromatographic purification of the crude product (column: 90 x 5.5 cm, silica gel 60 Merck, eluent: chloroform-methanol 10: 1) results in 10.62 g ($ 32.1 of theory) of powdered DC-pure N-Cbo -L- [3- (3,4-difyydroxy-phenyl) -2-methyl-alanyl] -L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine-methyl ester.

22.10 g (0.04 mole) of this ester is hydrolyzed and worked up as in Example 1, I with 232 ml of 1 N sodium hydroxide solution for 3½ hours at room temperature. 16.5 g (76.7% of theory) of DC-pure N-benzyloxy = carbonyl-L- [3- (3,4-dihydroxy-phenyl) -2-methyl-alanyl] -L-3- ( 3,4-dihydroxy-phenyl) -2-methyl-alanine (= N-Cbo-L-cc-methyl-dopyl-La-methyl-dopa).

The hydrogenolysis of the Cbo group is carried out as in Example 1, I listed.

1.25 g of palladium oxide is dehydrogenated in 100 ml of methanol and 30 ml of water. To this is added a solution of 13.46 g (0.025 mol) of N-Cbo-L-α-methyl-dopyl-L-α-methyl-dopa in 90 ml of methanol and hydrogenated for 3-4 hours. After processing, a brownish crude product is obtained, which is purified by column chromatography (column: 90 x 5.5 cm, silica gel 60 Merck; eluent: Acetone water 8: 1). The obtained DC pure eluate is evaporated in vacuo, taken up in 50 ml of water, treated with some carbon and dried in vacuo. 5.38 g of powdery analytically pure L- [3- (3,4-dihydroxy-phenyl) -2-methyl-alanyl] -L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine are obtained. (= La-methyl-dopyl-La-methyl-dopa), which, after drying in a vacuum desiccator, still contains 2.36 moles of water, [a] + 34.4 ° (c = 1, ).

The N-Cbo-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine used as the starting material can be prepared as follows.

Into a 1 liter three-neck flask equipped with a stirrer, 2 drip funnels and a gas line, 23.8 g of L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine (water content 11, 8%, MG 238). After complete removal of the air by means of a weak nitrogen flow, 150 ml of 2N sodium hydroxide solution are allowed to flow through the drip funnel and cool the solution to 0 ° C. During 25 minutes, a small solution of 66 ml of chloro-formic acid benzyl ester in 66 ml of two-cap and 150 ml of 2N sodium hydroxide solution is added dropwise with vigorous stirring and ice-cooling. After a further 30 minutes of stirring, slightly acidify with 6N hydrochloric acid and extract the reaction product 3 times with 200 ml of ether. The ether extract is shaken three times, each time with 50 ml of 0.5N hydrochloric acid and the ether solution is evaporated in vacuo after drying with sodium sulfate. 59.57 g (97.4% of theory) of N-benzyloxycarbonyl-L-3- (3,4-di-benzyloxycarbonyloxy-phenyl) -2-methyl-alanine are obtained as DC and analytically pure yellowish resin .

The splitting of the two O, O-Cbo groups is done with the apparatus set forth in Example I, I and the method of operation indicated. 12.26 g (0.02 mol) of N-benzyloxycarbonyl-L-3- (3,4-di-benzyloxycarbonyloxy-phenyl) -2-methyl-alanine are dissolved in 550 ml of THF and 890 ml of water and hydrolyzed with 160 ml of IN baking soda for one hour at room temperature. Then neutralize with approx. 160 ml of IN hydrochloric acid and completely distill THF in vacuo. After adding approx. 40 ml of water are taken up the crude hydrolysis product in 100 ml of vinegar ester and further worked up as described in Example 1, 5.80 g (84.196 of theory) of DC-pure N-Cbo-L-3- (3.4) are obtained. dihydroxy-phenyl) -2-methyl-alanine as a strong viscous yellowish oil.

Example 11.

L-alanyl-L-alanyl-L-5- (3,4-dihydroxy-phenyl) -2-methyl-alanine.

II, 25 g (0.05 mole) of L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine-methyl ester and 16.18 g (0.055 mole) of Cbo-L-alanyl-L- alanine (m.p. 152 -153 ° C, [oc] p -34.5 ° (c = 1, in methanol)) is subjected, as described in Example 1, to a peptide synthesis in 250 ml of dioxane with 10.3 g (0, 05 mol) DCC and worked up and purified. 11.76 (46.9% of theory) is obtained in powdered DC-pure Cbo-L-alanyl-L-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester.

12.5 g (0.025 mol) of Cbo-L-alanyl-L-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine methyl ester are hydrolyzed as described in Example 1, with 190 ml 0.5 N baking soda for 50 minutes at room temperature and work up. Yield 4.90 g (40.3% of theory) of DC-pure N-Cbo-L-alanyl-L-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine.

The hydrogenolysis of the Cbo group takes place analogously to that of Example 1, I. 1.0 g of palladium oxide is dehydrogenated in 50 ml of methanol and 5 ml of water. To this is added a solution of 9.75 g (0.02 mole) of N-Cbo tripeptide in 80 ml of methanol and hydrogenated for 3-4 hours. The colorless crude product is purified column chromatographically (column: 90 x 3.5 cm, silica gel 60 Merck; eluent: acetone-water 4: 1). The DC pure eluate is evaporated in vacuo and dried in desiccator. 6.14 g of analyzer are obtained L-alanyl-L-alanyl-L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine as a yellow powder containing 1.78 moles of water, [α] 0 + 20.5 ° (c = 1, in methanol).

Claims (1)

Analogous Process for Preparation of L-3- (3,4-Dihydroxy-phenyl} -2-methyl-alanine Peptides of the General Formula I HO \ CH3 H0 / V-CH2-C-C00H (i) NH -CQ-A-NH-R wherein A represents a straight or branched alkylene chain having 1 to 5 carbon atoms which may be substituted by a hydroxy group or with an optionally 1-2 hydroxy groups bearing phenyl group and R represents a hydrogen atom, an alkyl group with 1-3 carbon atoms, a glycyl group or an alanyl group, or pharmacologically acceptable salts thereof, characterized by condensing L-3- (3,4-dihydroxy-phenyl) -2-methyl-alanine of formula II HO HO-ft \ - CH2-C-C00H (II) x == y NH2 whose hydroxy groups and / or carboxy group may optionally be temporarily protected, with carboxylic acids of general formula III HOOC-AZ (III) or with reactive derivatives thereof A has the above meaning and Z means a reactive group or the group -NHR wherein R has the meaning given above, whereby if desired a hydroxy group present in the alkylene group A as well as the -NHR group may be temporarily protected, and in the event that Z means a reactive group, it is then replaced by the grouping -NHR and protecting groups